A mammary tumor model was developed in female Sprague-Dawley rats with the heterocyclic amine (HA) food carcinogen 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP). The effect of dietary fat on mammary tumorigenicity was also examined. The results show that a high fat diet caused more than a twofold increase in both mammary tumor incidence and average tumor mass observed in animals given PhIP. Histological analysis revealed that tumors from the PhIP-dosed rats maintained on the low fat diet were histologically benign whereas approximately 80% of the tumors from PhIP-treated rats on the high fat diet were histologically malignant. The data indicate that a high fat diet in combination with a HA found in cooked meat may enhance the incidence and severity of mammary gland cancer. Studies are also underway to examine the metabolic and molecular mechanisms by which PhIP induces mammary gland cancer and the mechanisms by which dietary fat accelerates this process. These studies focus on two major areas: (1) The analysis of genetic alterations in mammary tumors, and (2) analysis of the metabolic processing and DNA adduct formation of PhIP in the mammary gland. Changes in gene expression and genetic alterations in the tumors are being examined in order to assess the critical genes involved in HA-induced mammary carcinogenesis. Genes under study include neu, p53, c-myc, c-HA-ras, TGFalpha, and EGF receptor. Using single strand conformational polymorphism (SSCP) and direct sequencing of tumor DNA, no mutations have yet been detected in c-HA-ras and p53. Reverse transcriptase-polymerase chain reaction (RT-PCR) methods, developed to examine changes in tumor gene expression, revealed a high frequency of neu overexpression. Metabolic processing of PhIP is critical for PhIP-DNA adduct formation and initiation of carcinogenesis. PhIP-DNA adduct formation in the mammary epithelial cells, the target site for mammary cancer induction was examined at various times after a carcinogenic dose regimen of PhIP. PhIP-DNA adducts were present in the epithelial cells for at least six weeks after dosing. Adduct removal appeared to be slower when rats were on a high fat diet. The high PhIP-DNA adduct levels in the mammary gland of the rat appear to be associated with high phase II O-acetylation capacity of the mammary gland.